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Respiratory Physiology & Neurobiology 174 (2010) 135–145

Contents lists available at ScienceDirect

Respiratory Physiology & Neurobiology

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ffect of baroreceptor stimulation on the respiratory pattern: Insights intoespiratory–sympathetic interactions�

avid M. Baekeya,∗,1, Yaroslav I. Molkovb,1, Julian F.R. Patonc, Ilya A. Rybakb, Thomas E. Dicka,d

Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, USADepartment of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA 19129, USASchool of Physiology and Pharmacology, Bristol Heart Institute, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UKDepartment of Neurosciences, Case Western Reserve University, Cleveland, OH 44106, USA

r t i c l e i n f o

rticle history:ccepted 7 September 2010

eywords:eural control of respirationympathetic baroreceptor reflex

a b s t r a c t

Sympathetic nerve activity (SNA) is modulated by respiratory activity which indicates the existence ofdirect interactions between the respiratory and sympathetic networks within the brainstem. Our exper-imental studies reveal that Te prolongation evoked by baroreceptor stimulation varies with respiratoryphase and depends on the pons. We speculate that the sympathetic baroreceptor reflex, providing neg-ative feedback from baroreceptors to the rostral ventrolateral medulla and SNA, has two pathways:

n situ preparationomputational modelling

one direct and independent of the respiratory–sympathetic interactions and the other operating via therespiratory pattern generator and is hence dependent on the respiratory modulation of SNA. Our exper-imental studies in the perfused in situ rat preparation and complementary computational modellingstudies support the hypothesis that baroreceptor activation during expiration prolongs the Te via tran-sient activation of post-inspiratory and inhibition of augmenting expiratory neurones of the BötzingerComplex (BötC). We propose that these BötC neurones are also involved in the respiratory modulation

the r

of SNA, and contribute to

. Introduction

Cardiac, sympathetic and respiratory activities are coordinatedor effective and efficient gas exchange (Hayano et al., 1996). Inhis regard, the cardiovascular and respiratory systems can be envi-ioned as an integrated physiological system delivering oxygen tohe tissues and removing carbon dioxide from the body (Koepchent al., 1981; Richter and Spyer, 1990; Simms et al., 2010). The neuralontrol of this system is located within the brainstem where res-iratory rhythm and sympathetic premotor activity are generated.oreover, the respiratory and sympathetic control circuits inter-

ct within the brainstem and various sensory afferents, including

aroreceptor inputs, affect the neurones that generate and modu-

ate both respiratory and sympathetic activities.Sympathetic nerve activity (SNA) expresses respiratory modu-

ation even after vagotomy and decerebration (Barman and Gebber,

� This paper is part of a special issue entitled “Central cardiorespiratory regulation:hysiology and pathology”, guest-edited by Thomas E. Dick and Paul M. Pilowsky.∗ Corresponding author at: Division of Pulmonary, Critical Care and Sleepedicine, Department of Medicine, Case Western Reserve University School ofedicine, 11000 Euclid Avenue, Room 622 – Mailstop 5067, Cleveland, OH 44106-

067, USA. Tel.: +1 216 368 3964; fax: +1 216 368 0034.E-mail address: david.baekey@case.edu (D.M. Baekey).

1 These authors contributed equally to this work.

569-9048/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.resp.2010.09.006

espiratory modulation of the sympathetic baroreceptor reflex.© 2010 Elsevier B.V. All rights reserved.

1980; Haselton and Guyenet, 1989; Häbler et al., 1994; Richter andSpyer, 1990; Simms et al., 2009) supporting the idea of centralcoupling between respiratory and sympathetic networks. This cou-pling seems to be an important mechanism to: (i) optimize minuteventilation and cardiac output to increase the efficiency of oxygenuptake/perfusion at rest; and (ii) allow appropriate dynamic inte-grative cardiovascular and respiratory reflex responses essential formaintaining homeostasis (Hayano et al., 1996; Zoccal et al., 2009).Therefore, respiratory modulation may contribute to the dynamiccontrol of SNA.

The respiratory rhythm and motor pattern is generated by arespiratory central pattern generator (CPG) located in the lowerbrainstem (Bianchi et al., 1995; Cohen, 1979; Lumsden, 1923). Thepre-Bötzinger Complex (pre-BötC) located within the medullaryventrolateral respiratory column (VRC) is considered a majorsource of rhythmic inspiratory activity (Feldman and Del Negro,2006; Koshiya and Smith, 1999; Rekling and Feldman, 1998; Smithet al., 1991). The pre-BötC, interacting with the adjacent BötzingerComplex (BötC) containing mostly expiratory neurones (Ezure,1990; Ezure et al., 2003; Jiang and Lipski, 1990; Tian et al., 1999),

represents a core of the respiratory CPG (Bianchi et al., 1995; Cohen,1979; Richter, 1996; Richter and Spyer, 2001; Rybak et al., 2004,2007, 2008; Smith et al., 2007, 2009; Tian et al., 1999). This corecircuitry generates primary respiratory oscillations defined by theintrinsic biophysical properties of respiratory neurones involved,

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36 D.M. Baekey et al. / Respiratory Physi

he architecture of network interactions between respiratory neu-al populations within the pre-BötC and BötC, and inputs fromther brainstem compartments, including the pons, retrotrapezoiducleus (RTN), raphé, and nucleus tractus solitarii (NTS).

The NTS receives and integrates most of the peripheral cardio-ascular and respiratory afferent inputs, including baroreceptorfferents. It mediates a variety of reflexive motor responses fromhe brainstem and spinal cord that provide homeodynamic controlf breathing and cardiovascular functions (reviewed by Loewy andpyer, 1990). The classical baroreflex control of SNA operates viaTS neurones mediating the baroreceptor projections to the cau-al ventrolateral medulla (CVLM). Through this path, baroreceptorctivation excites CVLM neurones which in turn inhibit the ros-ral ventrolateral medulla (RVLM) neurones and hence lower SNADampney, 1994; Guyenet, 1990). This pathway provides a directegative feedback control of SNA.

Respiratory activity is also modulated by the afferent barorecep-or activity possibly through the same NTS baro-sensitive neurones.or instance in vagally intact, decerebrate cats, the activities ofpproximately 50% of the respiratory-modulated neurones withinhe VRC responded to transient pressure pulses (Dick and Morris,004). Furthermore, expiratory propriobulbar activity was mod-lated to a greater extent than inspiratory propriobulbar activityDick and Morris, 2004). Because the overlap between respira-ory and cardiovascular regulatory areas is extensive, we could notxclude the possibility that neurones with both the respiratory-odulated and arterial pulse-modulated activity were related to

ardiovascular rather than to respiratory control or to both. Toddress this, we restricted our analysis to respiratory-modulatedctivity that was identified as either bulbospinal premotor or bul-ar (laryngeal) motor neurones. Surprisingly, these neurones alsoxhibited arterial-pulse-pressure modulated activity (Dick et al.,005). Thus, neurones with an identified respiratory function, mod-lating airway resistance, can express arterial-pulse modulatedctivity.

The aim of this study was to verify that activation of barore-eptor afferents can indeed affect the respiratory CPG and theespiratory pattern by altering BötC expiratory neurone firing. Weerformed systematic recordings of respiratory neurones withinhe VRC, specifically within the BötC/pre-BötC core of the respira-ory network, to reveal possible neural interactions between thearo-afferent processing circuits and those determining the respi-atory pattern.

Another important objective of this study was to examine theossibility of parallel pathways for the sympathetic baroreceptoreflex. Parallel pathways, one respiratory modulated and the otherndependent of respiration has been proposed and identified forhe sympathetic chemo-reflex (Guyenet and Koshiya, 1995). Theheoretical basis for parallel pathways was derived from the fol-owing logic. If respiratory modulation represents an important

echanism for control of SNA and, at the same time, barorecep-or activation can alter the respiratory pattern, then baroreceptorctivation should affect SNA via its effects on the respiratory patternndependent of the direct baroreflex pathway from NTS to CVLM.his would provide a basis for suggesting that the sympatheticaroreceptor reflex provides negative feedback from baroreceptorso SNA through two pathways: one direct that is independent of theespiratory–sympathetic interactions, and the other indirect thatepends on the effects of baroreceptor afferents on the respiratoryattern and hence on the respiratory modulation of SNA.

Finally, because transection of the pons significantly attenu-

tes respiratory modulation of SNA (Baekey et al., 2008; Dick etl., 2009), we were interested in a possible role of the pons inhe baroreceptor reflex mediated respiratory–sympathetic interac-ions and respiratory modulation of SNA. We used a computational

odel of the brainstem respiratory network that includes the pons

& Neurobiology 174 (2010) 135–145

(Smith et al., 2007) and extended it to incorporate VLM, RTNcompartments and their corresponding neural populations. Theextended model was used to simulate, investigate and predict thepossible neural mechanisms involved in the baroreceptor reflexmediated respiratory–sympathetic interactions.

2. Materials and methods

2.1. Surgical and experimental procedures

This series of experiments were performed at two sites: theUniversity of Bristol in Bristol, England, and Case Western ReserveUniversity in Cleveland, OH, USA. For those experiments performedin England, all surgical and experimental procedures conformed tothe UK Animals (Scientific Procedures) Act and were approved bythe University of Bristol ethical review committee. Similarly, forthe experiments done in the United States, the Institutional AnimalCare and Use Committee (IACUC) of Case Western Reserve Univer-sity approved the protocols. The experiments were performed onjuvenile (P21–P28, 60–100 g) male rats; these rats were suppliedfrom a Wistar strain at the University of Bristol; whereas those atCase Western Reserve University were Sprague–Dawley.

The rat strain was the only major difference between the twostudies because the same individual (David Baekey) performed theexperiments using equipment from the same suppliers.

2.2. General surgical methods

We used the arterially perfused in situ preparation for theseexperiments (Paton, 1996). Animals were pretreated with hep-arin sodium (1000 units – IP), anesthetized with isoflurane (2–3%),and then bisected below the diaphragm. The cranium and thoraxwere submerged in cold artificial cerebrospinal fluid (aCSF), decere-brated, skinned, and eviscerated. The phrenic nerve and descendingaorta were dissected and the dorsal medullary surface exposed.

After this preliminary surgery, the preparation was mountedsupine in a stereotaxic frame in the recording chamber. Thedescending aorta was cannulated with a #4 French, double-lumencatheter (Braintree Scientific). Through one lumen, the preparationwas perfused with iso-osmotic aCSF saturated with 95% O2/5% CO2(21–28 ml/min, Marlow Watson 505S peristaltic pump). Throughthe other lumen perfusion pressure was monitored (CWE TA-100 transducer-amplifier). Perfusion pressure was maintained at60–80 mm Hg and supported with 4 �M vasopressin (20 �l addedto perfusate, basically to restore the vasopressin lost by removingthe blood and hypothalamus). The preparation was immobi-lized with vecuronium bromide (0.4 mg/200 ml perfusate). Sodiumcyanide (0.1%, 50 �l-bolus intra-arterial) was used to initiate rhyth-mic respiratory activity through robust, transient stimulation of thecarotid chemoreceptors.

2.3. Ponto-medullary transection

The left phrenic nerve and thoracic sympathetic nerves weredrawn into suction electrodes and efferent neural activities wereamplified (Grass P511), filtered (0.1–3 KHz) and digitized using aCED Power 1401 connected to a Dell Personal Computer runningCED Spike2 software.

After placement of the peripheral nerves in the recording elec-trodes and baseline recordings including the characterizing theresponses to transient increases in the perfusion pressure to stim-

ulate arterial baroreceptors, the pons was separated from themedulla (Baekey et al., 2008). Briefly, a razor blade trimmed to theappropriate width was lowered perpendicularly through the brain-stem approximately 3 mm rostral to calamus scriptorius (Baekeyet al., 2008). This transection resulted in a transient suppression of

ology & Neurobiology 174 (2010) 135–145 137

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Fig. 1. Response of the sympathetic (SNA, top pair of traces) and phrenic (PNA, mid-dle pair of traces) nerve activities to transient increases in perfusion pressure (PP,bottom trace). (A1–A3) Stimulation was applied to the intact preparation duringinspiration (A1), post-inspiration (A2) and late expiration (A3). Note that the stim-ulus applied during inspiration did not affect the respiratory pattern (A1), whereasthe stimuli delivered during the expiratory phase prolonged expiration (A2, A3).

D.M. Baekey et al. / Respiratory Physi

espiratory efferents followed by an unstable apneustic pattern. Ifhe respiratory rhythm needed to be restored, the percent of carbonioxide was increased in the aCSF for the duration of the recording.

After the post-transection baseline recording, perfusion pres-ure of the preparation was transiently increased for comparisonith the “intact” state (Fig. 1B).

.4. Electrode placement and neuronal sampling strategy

The multi-electrode array was secured to a stereotaxic framellowing the 16 tungsten microelectrodes (10–12 M�) to beligned perpendicularly to the dorsal medullary surface. Eightlectrodes were on either side of the brainstem in two rowsf four electrodes oriented sagittally. The two rows were sepa-ated 250 �m while electrodes within each row were separatedy 300 �m. Stereotaxic coordinates were used to position elec-rodes bilaterally in the rostral lateral medulla. The depth of eachlectrode was positioned in steps as small as a micron and thisdjustment was used to maximize signal-to-noise ratio and to iso-ate the recording of activity to a single source. In cases where morehan one neurone was recorded on a single electrode, the principleomponent analysis (PCA) feature of the Spike2 software was usedo discriminate individual spike trains (spike sorting). The inde-endent depth adjustment of each electrode optimized the yield ofarallel single neurone recordings.

.5. Data analysis

The recorded data include: phrenic nerve activity (PNA), tho-acic sympathetic nerve activity (tSNA) and extracellular potentialsrom the microelectrode array. PNA was processed by removal ofny DC offset, rectification and smoothing using a 100-ms timeonstant to obtain a moving-time average of activity. From thisintegrated’ PNA, we marked the onsets of inspiratory and expi-atory phases. Action potentials of single neurones were convertedo times of occurrence, i.e. spike trains (Fig. 5B).

The following measures were computed from a 10-min base-ine period in order to ensure that there was only one neuroneer channel and to characterize the cell-type: (1) Autocorrelationistograms were created for each spike train to ensure that it rep-esents the activity of a single neurone (not shown). A spike trainith potentials from two or more neurones would include short

ntervals not constrained by refractoriness. (2) Cycle-triggered his-ograms were used to classify activity patterns with significantespiratory modulation according to the phase (inspiratory or expi-atory) in which they are more active and by trends in their burstatterns (augmenting, decrementing or plateau). (3) The firing rateistograms were plotted (Fig. 5B – 100 ms bins) to examine the

nfluence of arterial pulse pressures on activity.

.6. Modelling and simulations

The model was developed based on, and as an extension of, therevious model described by Smith et al. (2007). All neurones wereodelled in the Hodgkin–Huxley style (single-compartment mod-

ls) and incorporated known biophysical properties and channelinetics characterized in respiratory neurones in vitro. Each neu-onal type was represented by a population of 20–50 neurones.eterogeneity of neurones within each population was set by a ran-om distribution of some parameters and the initial conditions for

alues of membrane potential, calcium concentrations, and channelonductances. A full description of the previous model and modelarameters can be found in Smith et al. (2007). All new (additional)nd altered (relative to Smith et al., 2007) model parameters arendicated in Table 1 in the Appendix.

This TE prolongation was stronger when stimulus was applied later in expiration.(B) The effect of baroreceptor stimulation after pontine transection. The appliedstimulus did not prolong expiration, but shortened the apneustic inspiratory PNAbursts. Traces from top to bottom: integrated and raw SNA, integrated and raw PNA,and perfusion pressure are shown on each panel.

1 ology & Neurobiology 174 (2010) 135–145

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Fig. 2. Conceptual model of interaction among respiratory-related activity of theventral respiratory column (VRC), pontine circuits (PONS), sensory network inthe nucleus tractus solitarii (NTS) and rostral and caudal ventrolateral medulla(RVLM/CVLM). The sympathetic baroreceptor reflex operates via two pathways(thick line, large arrows): one direct pathway includes baroreceptors, NTS baro-sensitive cells and CVLM, which inhibits RVLM and SNA; the other indirect pathwaygoes from baroreceptors through NTS and VRC, whose post-inspiratory neuronesinhibit RVLM and SNA. Grey arrows represent the effects of VRC and PONS on RVLMproviding respiratory modulation of SNA. Another grey arrow indicates the effectsof VRC on PONS providing respiratory modulation of pontine neurones. Finally, two

38 D.M. Baekey et al. / Respiratory Physi

All simulations were performed with a simulation package NSM.0, developed at Drexel University by S.N. Markin, I.A. Rybak, and.A. Shevtsova. Differential equations were solved using the expo-ential Euler integration method with a step of 0.1 ms. Other detailsf modelling and simulation methods can be found in Smith et al.2007).

. Results

.1. Respiratory phase-dependent and pontine-dependent effectsf transient baroreceptor stimulation on the respiratory patternnd on respiratory modulation of sympathetic nerve activity

In the intact arterially perfused in situ rat preparation, thehoracic sympathetic nerve activity (tSNA) usually exhibited

well-expressed positive inspiratory modulation reaching itseak in early post-inspiration (see Fig. 1A1–A3 before appliedtimulations). The respiratory modulation of tSNA was signifi-antly suppressed or eliminated after removal of the pons whenhrenic nerve activity (PNA) transformed to a more apneustic-

ike pattern with prolonged inspiratory bursts and shortenedxpiration periods (see Fig. 1B before applied stimulation) henceonfirming a critical role of the pons for the observed respira-ory modulation of tSNA as reported previously (Baekey et al.,008).

The transient increases in the perfusion pressure (PP) werenduced by increases in flow of perfusate with the amplitudelightly above the threshold for evoking the sympathetic barore-ex (Baekey et al., 2008; Dick et al., 2009; Simms et al., 2007). Thesetimuli were delivered during inspiration, post-inspiration or latexpiration and produced an obvious respiratory phase-dependentffect on the respiratory pattern (evaluated by phrenic nerve activ-ty, PNA) and, correspondingly, on the respiratory modulation ofSNA (Fig. 1A1–A3).

With pons intact, the barostimulation had almost no effectn the amplitude and duration (i.e. inspiratory period, TI) of thehrenic bursts even when stimuli were delivered during inspirationFig. 1A1, n = 8 animals). At the same time, these stimuli suppressedr abolished respiratory modulation of tSNA. The baroreflex in allhese cases persisted, i.e. tSNA was suppressed during barostimu-ation. In contrast, the same stimuli applied during post-inspirationnd late expiration produced an increase in the expiration periodTE) and decrease in tSNA. The barostimulation-evoked prolonga-ion of TE was greater if stimulation was applied later during thexpiratory phase. For example, Fig. 1A2 and A3 shows a greaterncrease in TE when stimulation was delivered later in expirationpanel A3), than when it was applied during post-inspiration (panel2).

As mentioned above and previously (Baekey et al., 2008; Dick etl., 2009), after pontine transection the respiratory modulation ofSNA was greatly reduced. However, the baroreflex persisted (low-ring tSNA during baroreceptor stimulation). Further, when theerfusion pressure pulse was applied during the prolonged inspi-ation, the barostimulation shortened the apneustic burst (Fig. 1B),hich is stark contrast to a negligible effect on Ti in the intact state

Fig. 1A1).

.2. Conceptual model and hypotheses

Fig. 2 represents our conceptual schematic of respiratory–

ympathetic interactions in the context of the sympathetic barore-eptor reflex. The schematic is based in part on our experimentaltudies described above and previously (Baekey et al., 2008; Dickt al., 2009), as well as on various data in the literature. Specifi-ally, we hypothesize that sympathetic baroreflex operates via two

dashed arrows indicate central suppression of the baroreflex gain during inspira-tion which potentially can be provided by VRC or pontine neurones see details inthe text).

pathways (see solid black arrows in Fig. 2). The first path leads fromNTS to the CVLM, which in turn inhibits RVLM neurones hencelowering sympathetic motor output (Dampney, 1994; Guyenet,1990). This pathway avoids respiratory circuits and provides neg-ative feedback control of SNA independent of the respiratoryactivity.

The second pathway is mediated by respiratory circuitsin the VRC and pons and represents a baroreflex compo-nent dependent on the respiratory–sympathetic interactions.As mentioned above, the respiratory modulation of sympa-thetic activity includes inspiratory activation and post-inspiratoryinhibition or disfacilitation (Fig. 1A1–A3). Because both thesemodulatory influences require the pons to be intact for theirexpression (Fig. 1B), and because baroreceptor stimulationin the intact preparation prolongs expiration (Fig. 1A2 and A3),we hypothesize that the respiratory modulation of SNA is mainlyprovided by two types of connections to the RVLM (grey arrowsin Fig. 2): (1) a direct excitatory connection from pontine neu-rones whose activity increases during inspiration peaking duringthe inspiratory-to-expiratory phase transition (IE neurones), pro-viding the positive inspiratory and IE phase transition modulationof RVLM neurones and SNA; and (2) inhibitory connections fromthe post-inspiratory neurones of the VRC, whose activity criticallydepends on pontine drive (Dutschmann and Herbert, 2006; Rybaket al., 2004; Smith et al., 2007) and is controlled by inputs frombaroresponsive RTN neurones (see above). The latter connectionsprovide the negative post-inspiratory modulation of RVLM neu-rones and SNA. Therefore, we specifically hypothesize that (withpons intact) the second baroreflex pathway operates via the acti-vation of VRC’s post-I neurones (Fig. 2) that prolong expiration andinhibit RVLM neurones and SNA (Fig. 1A2 and A3).

The respiratory modulation of pontine neurones, includinginspiratory and phase-spanning inspiratory–expiratory types, isprovided by various projections of VRC’s respiratory neurones tothe corresponding pontine neurones (indicated by the grey arrow

from VRC to pons in Fig. 2; see Ezure and Tanaka, 2006; Rybak etal., 2004, 2008; Segers et al., 2008).

Our experimental data (Fig. 1A1) also suggest that when thepons is intact the effect of baroreceptor stimulation on the res-

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iratory rhythm is minimal during inspiration implying that theTS barosensitive interneurones that excite post-I neurones ofRC are suppressed during inspiratory phase. In contrast, short-ning of the inspiratory (PNA) bursts by baroreceptor stimulations observed after pontine removal (Fig. 1B). Hence, we hypoth-size the existence of inspiratory inhibition (gain reduction) ofhe barosensitive NTS neurones during inspiration (dashed arrowsn Fig. 2), which can potentially be produced by either pon-ine (inspiratory modulated) neurones (Felder and Mifflin, 1988;otter, 1981), or inhibitory inspiratory neurones of VRC (e.g.ee Miyazaki et al., 1999), whose activity depends on pontinerive.

.3. Computational modelling

One of the objectives of this study was to investigate the aboveonceptual model and hypotheses proposed using computationalodelling. We used the previously published large-scale compu-

ational model of the brainstem respiratory network (Smith etl., 2007) and extended this model by incorporating NTS, RVLM,VLM and lateral pontine compartments (see Fig. 3). The basicodel described interactions among respiratory neurone popula-

ions spatially organized within the lateral brainstem, extendingrom the pons to the lower medulla. Similar to that model, the res-iratory brainstem populations in the extended model (see Fig. 3)

nclude (right-to-left): a ramp-inspiratory (ramp-I) population ofremotor bulbospinal inspiratory neurones and an inhibitory early-

nspiratory (early-I(2)) population located in the rostral ventralespiratory group (rVRG); a pre-inspiratory/inspiratory (pre-I/I)nd an inhibitory early-inspiratory (early-I(1)) populations of there-Bötzinger Complex (pre-BötC), and an inhibitory augmenting-xpiratory (aug-E) and a post-inspiratory (post-I) populations and

n excitatory pre-motor post-I(e) populations in the Bötzingeromplex (BötC). The BötC’s and pre-BötC’s populations togetherepresent a core circuitry of the respiratory CPG (Rybak et al.,007; Smith et al., 2007). In addition, multiple drives from otherrainstem components, including the pons, RTN, and raphé pro-

ig. 3. The schematic of the extended computational model showing interactions betweeents involved in the control of breathing and sympathetic motor activity pons, RTN, Böhich consists of 20–50 single-compartment neurones described in the Hodgkin–Huxl

dditionally incorporates RVLM and CVLM populations in the VLM, an IE phase-spanningodel includes three sources of tonic excitatory drive located in the pons, RTN and raphé

ources project to multiple neural populations in the model (green arrows). However, to siarticular populations. The full structure of connections from each drive source (pons/RTeights are in Table 1 in the Appendix.

& Neurobiology 174 (2010) 135–145 139

vide excitatory drives that regulate the dynamic behaviour ofthis core circuitry, as well as the activity of premotor neuronepopulations in the rVRG and phrenic motor output (PN) [noteto distinguish the model’s data from the experimental record-ings we have purposefully introduce new abbreviations to PNrather than PNA and SN rather than tSNA]. The respiratoryoscillations in the model emerge within the BötC/pre-BötC corecircuitry due to dynamic interactions between (i) the excitatoryneural population in pre-BötC active during inspiration (pre-I/I),(ii) the inhibitory population in pre-BötC that provides inspi-ratory inhibition within the network (early-I(1)); and (iii) theinhibitory populations in the BötC generating expiratory inhi-bition (post-I and aug-E) (see Rybak et al., 2007; Smith et al.,2007).

To extend this model, we have incorporated the following newneural populations (see Fig. 3): (1) RVLM and CVLM populationscomprising the ventrolateral medulla (VLM) compartment; (2) apontine IE neural population; and (3) two identical neural popu-lations of second-order barosensitive cells in the NTS. Similar toother populations in the model, each of these populations con-tains 20–50 neurones with randomized parameters modelled inthe Hodgkin–Huxley style (single-compartment models). The neu-rones in these populations contain only a minimal set of ionchannels necessary for spike generation (fast sodium, potassiumrectifier and leakage) with the same parameters as those used forother neurones of this type in the basic model (e.g., ramp-I neu-rones, see Smith et al., 2007).

The RVLM and CVLM populations were added as these compart-ments are considered critical for sympathetic output (Dampney,1994; Guyenet, 2000). Neurones with phase-spanning IE pat-tern and the peak of the activity at the end of inspiration havebeen found in the rostral pons (Cohen and Wang, 1959; Dick

et al., 1994, 2008; Segers et al., 2008; Takagi and Nakayama,1958). The two populations of barosensitive neurones in the NTSreceive “barostimulation” in our simulations. The applied stimuliwere simulated as excitatory synaptic drive to these popula-tions with time-dependent function reproducing the dynamics

n different populations of respiratory neurones within major brainstem compart-tC, pre-BötC, rVRG, VLM, Raphé, and NTS). A ‘sphere’ represents each population,

ey style. In comparison with the previous model (Smith et al., 2007), this modelpopulation in the pons, and two populations of barosensitive cells in the NTS. Theshown as green triangles. These drives, especially those from the pontine and RTN

mplify the schematic, only the most important connections are shown connected toN/raphé) to target neural populations of the model and the corresponding synaptic

140 D.M. Baekey et al. / Respiratory Physiology

Fig. 4. Simulations of the effect of phase-dependent barostimulation on the phrenic(PN) and sympathetic (SN) nerve outputs. Stimulus was applied during inspira-tion (A1), post-inspiration (A2) and late expiration (A3). In A1, the stimulus appliedduring inspiration had no effect on respiratory phase durations; SN activity duringstimulation is attenuated due to baroreflex. In (A2, A3), the stimuli during expira-tion prolonged expiration, slightly when applied in post-inspiration or substantially,in late expiration. (B) After removal of the pontine compartment in the model,the applied stimulation does not prolong the expiratory period, but shorten the“apneustic” PN bursts if applied during inspiration. SN activity respiratory modu-la

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contrast, stimuli applied during late expiration (Fig. 4A3) prolong

ation is abolished. Traces are as Fig. 1. The phases of barostimulation are chosenpproximately the same as in experimental examples in Fig. 1.

f PP in the corresponding experiments (see bottom traces inigs. 4 and 5A).

The following connections between the incorporated popula-ions described above and other neural populations have beenncluded in the model (Fig. 3; see also Table 1 in the Appendix):

1) Excitatory connections from the RVLM population to thesympathetic preganglionic neurones (IML), which define thesympathetic motor output (SNA; Dampney, 1994; Guyenet,

2000).

2) Inhibitory connections from the CVLM to the RVLM pop-ulation and excitatory connections from the population ofsecond-order barosensitive neurones in NTS to the inhibitory

& Neurobiology 174 (2010) 135–145

CVLM population which represent the classical, direct path-way of sympathetic baroreflex (Guyenet, 2000; Mandel andSchreihofer, 2006, 2009).

(3) Excitatory connections from the ramp-I population of rVRG andfrom post-I(e) population in BötC to the pontine IE populationwhich are proposed to form the phase-spanning IE pattern ofthe latter (see Rybak et al., 2004);

(4) Excitatory connections from the pontine IE population to theRVLM population, which we specifically hypothesize in thisstudy to provide the pontine-dependent inspiratory and post-inspiratory modulation of SNA (see Fig. 1A1–A3).

(5) Inhibitory connections from the post-I population of BötC to theRVLM population, which we hypothesize to explain the neg-ative post-inspiratory modulation of SNA (Fig. 1A1–A3). Thelatter modulation is pontine-dependent, since in our modelpontine drive is necessary for the expression of post-I activity(see Rybak et al., 2007; Smith et al., 2007). Note that direct con-nections from the BötC region to RVLM have been previouslyexperimentally identified by Sun et al. (1997).

(6) Excitatory connections from one population of second-orderbarosensitive neurones in the NTS to the post-I population ofBötC, which is hypothetically responsible for the prolongationof expiration during baroreceptor stimulation (Fig. 1A2 and A3).

(7) Finally, we put a hypothetical inhibitory connection fromthe pontine-dependent early-I(2) population of rVRG to thesecond-order barosensitive NTS population projecting to thepost-I neurones to control (reduce) the gain of barostimulationon the respiratory pattern during inspiration. This central con-trol of NTS cells during inspiration has been demonstrated forthe NTS pump cells by Miyazaki et al. (1999) and P cells doreceive baro-receptor input (Rogers et al., 1993). This centralcontrol of baroreflex gain in the model is pontine-dependent,since in our model pontine drive is necessary for the expressionof activity in the early-I(2) population (see Rybak et al., 2007;Smith et al., 2007).

Fig. 4 shows the results of our simulation of the effects of tran-sient barostimulation during different phases of the respiratorycycle using the “intact” model (Fig. 4A1–A3) and after removal ofthe pontine compartment (Fig. 4B). Under normal conditions (i.e.with pons intact and in the absence of “baroreceptor stimulation”)the model generates a three-phase eupnoea-like respiratory pat-tern and augmenting PN bursts similar to those observed in thein situ preparations under normal conditions (see PN output inFig. 4A1–A3, all details can be found in Smith et al., 2007). Sim-ilar to our experimental records (Fig. 1A1–A3), the sympatheticoutput (SN) in the model exhibits a positive inspiratory and post-inspiratory modulation provided by the pontine IE population toRVLM and a rapid-offset during post-inspiratory resulting from theinhibitory inputs from the post-I population of BötC to the RVLM(see Figs. 3 and 4A1–A3). Transient barostimulation applied tothe barosensitive NTS populations produces a temporal reductionof sympathetic output via direct activation of the CVLM popula-tion that inhibits the activity of RVLM population. This representsthe direct, respiratory-independent component of the sympatheticbaroreflex.

Baroreceptor stimulus application during inspiration does notaffect respiratory (PN) activity in the model because the gain of theinput from the barosensitive NTS population to the post-I neuronesis suppressed centrally during inspiration by the inhibitory early-I(2) population of rVRG (see Fig. 4A1 and compare with Fig. 1A1). In

expiration via activation of post-I neurones of BötC, which inhibitthe aug-E population and the RVLM. These interactions represent asecond component of the sympathetic baroreflex involving interac-tions between the respiratory and sympathetic circuits. Note that,

D.M. Baekey et al. / Respiratory Physiology & Neurobiology 174 (2010) 135–145 141

Fig. 5. Response to transient baroreceptor stimulation in the model and in situ. (A) Simulation results. The top pair of traces: membrane potential of a randomly chosenneurone from the post-I population of BötC and integrated spike histogram of the entire post-I population. Second pair of traces: membrane potential of a randomly chosenneurone from the aug-E population of BötC and spike histogram of the entire aug-E population. The applied stimulation (lowest trace) excited post-I neurones and inhibitedaug-E neurones. After the stimulus ended, the aug-E neuroneactivated again. The transient changes in post-I and aug-E activities prolonged expiration. (B) Experimentalresults in situ preparation: the applied baro-stimulus activated post-I and suppressed aug-E activities. These changes in neurone activity were associated with decreasedSNA output and prolongation of expiration. Traces in (B): top two traces show an extracellular recording from a post-I neurone in BötC and the histogram of its activity; thenext pair of traces show a simultaneous extracellular recording from an aug-E neurone of BötC and the corresponding histogram; the remaining traces show the integratedphrenic nerve activity (PNA), integrated thoracic sympathetic nerve activity (tSNA), and perfusion pressure (PP). The response to the applied barostimulation in the in situp d prop these

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reparation is associated with increases in post-I and decreases in aug-E activity anost-I and aug-E neurones are consistent with the proposed connectivity between

imilar to our experimental data (Fig. 1A3), stimulation-evokedrolongation of expiration is greater if stimulation is applied lateruring the expiratory phase (Fig. 4A2 and A3).

Removing the pontine compartment in the model convertshe normal eupnoea-like respiratory pattern to the apneustic pat-ern characterized by prolonged PN busts with a rectangle-likerofile (see Fig. 4B). As shown previously (Rybak et al., 2007;

mith et al., 2007), this pattern is characterized by a lack of post-activity that is strongly dependent on pontine drive. With the

emoval of the pons in the model, the respiratory modulation ofNA (formed by inputs from the IE pontine population and theötC’s post-I population to RVLM) is abolished (see Fig. 4B and

longation of the expiratory phase. These neuronal activity patterns of the recordedtwo types of neurones located in the BötC.

compare with Fig. 1B). Simultaneously, the central suppressionof the baroreflex gain by the rVRG’s early-I(2) population, whoseactivity in the model is also dependent on the pontine drive, iseliminated with the pontine removal. Therefore the applied “baros-timulation” can activate post-I population during inspiration andproduce an advanced termination of the apneustic inspiratorybursts hence shortening inspiration (see Fig. 4B and compare with

Fig. 1B).

Fig. 5A illustrates the neural mechanism by which the transientbarostimulation applied during expiration prolongs this expira-tion in the intact model. This simulation corresponds to thecase shown in Fig. 4A3. The post-I neurones of BötC when acti-

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42 D.M. Baekey et al. / Respiratory Physi

ated inhibit all inspiratory (and aug-E) neurones and initiate theost-inspiratory phase of expiration. During expiration these neu-ones adapt hence releasing activity of the aug-E neurones fromnhibition allowing for their gradual activation (see unperturbedreathing cycles in Fig. 5A). When a barostimulus comes duringxpiration, the post-I population is activated again and inhibitshe aug-E population, hence producing a “resetting of expira-ion”. The aug-E population then is activated second time. Thisesetting of expiration by the transient barostimulation applieduring expiration results in the observed prolongation of expirationuration.

.4. Transient pressure pulses delivered during expiration prolonghis expiration via activation of post-I neurones of the Bötzingeromplex in situ

Given the consistent prolongation of TE in response to tran-ient pressure pulses (see Fig. 1A2 and A3) and our modellingredictions (Fig. 5A), we investigated the responses of expiratoryeurones of BötC to baroreceptor stimulation. Preliminary experi-ents (n = 4 animals) were performed using the arterially perfused

n situ preparations of juvenile rats. Extracellular recordings wereade from neurones within the region extending 1.7–2.7 mm ros-

ral to the obex, from 1.2 to 2.2 mm lateral to the midline, andentrally ∼2.6 mm below the dorsal medullary surface. This areaorresponded to the BötC in the rat (Paxinos and Watson, 1998.).

e focused on determining the response to barostimulation onxpiratory decrementing (post-I) and expiratory augmenting (aug-) neurones (see example in Fig. 5B). We recorded decrementingpost-I type (n = 7) and augmenting expiratory neurones with theatter augmenting throughout the phase (aug-E type, n = 6). Whenerfusion pressure was increased transiently, the activity of post-Ieurones increased (n = 5 of 7) and the activity of aug-E neuronesecreased (n = 6 of 6) in full accordance with the model predic-ion (see example shown in Fig. 5B and compare with Fig. 5A).his pattern of activation/deactivation of post-I and aug-E cells,espectively, was consistent with the reciprocal inhibitory con-ections between the post-I and aug-E neurones in the BötC (seeig. 3). In the majority of cases (n = 15 of 25), the barostimulationpplied during expiration resulted in the prolongation of TE, andhis prolongation was greater when stimulation was applied later inxpiration.

. Discussion

.1. Respiratory modulation of sympathetic activity

In this study, we focus on two aspects of the respiratory modu-ation of SNA: the positive inspiratory (or IE with the peak duringarly post-inspiration) and the subsequent post-inspiration rapidff-set that can be recognized in our experimental recordings initu (Fig. 1A1–A3). Our data support the anatomical evidence thatympatho-respiratory coupling occurs, at least partially, in theedulla (Pilowsky et al., 1990, 1992, 1994; Sun et al., 1997). How-

ver, recordings from RVLM and CVLM neurones have revealedore complex respiratory-modulated activity patterns supporting

roader interactions between the brainstem respiratory neuronesnd neurones within RVLM and CVLM than we have developedere. For example, Mandel and Schreihofer (2006) revealed at

east four distinct respiratory modulated discharge patterns: inspi-atory modulated activity with a peak during PNA; expiratory

odulated activity decreasing during PNA; expiratory–inspiratoryodulated activity, and post-inspiratory modulated activity. These

our types of respiratory-modulated activity were established usingycle-triggered histograms of neuronal activity correlated to cycleriggered averages of integrated phrenic nerve activity (PNA). With

& Neurobiology 174 (2010) 135–145

our initial model, we have reproduced aspects of the respiratory-modulated SNA pattern without all the respiratory-modulatedactivities expressed in RVLM/CVLM. This issue requires furtherdevelopment of the model.

While our model does not contain connections betweenthe respiratory pattern generator and neurones in CVLM, itreplicates major features of sympatho-respiratory patterning.Additional experimental data are needed to elaborate, validateor revise the model. Specifically, another possibility is that therespiratory modulation of RVLM neurones is related throughCVLM.

According to our previous (Baekey et al., 2008; Dick et al.,2009) and current studies, the magnitude of respiratory modulationof SNA depends on the pons being intact (Fig. 1B). After ponto-medullary transection, respiratory modulation is only weakly seenin thoracic sympathetic nerve activity. This reduced couplingof phrenic and sympathetic nerve activities following ponto-medullary transection is similar to that of the decerebrate ratafter systemic administration of MK-801, an NMDA antagonist(Morrison, 1996). Further, the effects of MK-801 on the respiratorypattern (decreased amplitude, apneusis, increased variability) mir-ror those of ponto-medullary transection (Lumsden, 1923; Smith etal., 2007). The significant reduction of SNA respiratory modulationwith removal or suppression of the pons suggests that RVLM/CVLMneurones defining the SNA profile either receive direct projectionsfrom respiratory modulated pontine neurones or receive inputsfrom the respiratory neurones in VRC whose activity criticallydepends on pontine input. Thus, the respiratory modulation of SNAcould be mediated directly by pontine IE neurones and/or indirectlyby BötC’s post-I neurones, whose activity was, in part, pontine-dependent. This scenario was implemented in our model. Otherpossible solutions will be comparatively considered in our futurestudies.

4.2. Effects of baroreceptor stimulation on the respiratory patternand the activity of respiratory neurones

Our data provide a neural substrate for the expiratory–facilitatory response to activation of baroreceptors (Brunner et al.,1982; Dove and Katona, 1985; Grunstein et al., 1975; Li et al.,1999a,b; Lindsey et al., 1998; Nishino and Honda, 1982; Richterand Seller, 1975; Speck and Webber, 1983; Stella et al., 2001).We clearly demonstrate that barostimulation activates post-I neu-rones and depresses aug-E neurones. This is consistent with ourprevious finding that respiratory neurones, preferentially expira-tory neurones, are modulated with the arterial pulse (Dick andMorris, 2004; Dick et al., 2005). In contrast, these findings differfrom those of Kanjhan et al. (1995) who did not find barosensitiveneurones in the BötC and to McMullan et al. (2009) who found onlya modest increase in TE evoked by aortic depressor nerve (ADN)stimulation.

Our model replicates prolongation of expiration and iden-tifies possible network interactions following baroreceptoractivation.

The prolongation of expiration in response to abrupt increasesin blood pressure does not appear to have a teleological explana-tion. On the other hand the inverse, volitional slowing of breathingor Pranayamic breathing is a recognized practice that decreasesblood pressure. The mechanism is unclear but prolonged expira-tion may increase vagal tone (to decrease heart rate) and decreasevenous return (temporary cessation of abdominal thoracic pump-

ing), vascular resistance and cardiac work.

A prolongation of expiratory period through activation ofpost-I neurones also occurs in the diving response (Dutschmannand Paton, 2002). This evokes an increase in cardiac vagaltone (bradycardia), systemic vasoconstriction as well as an

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D.M. Baekey et al. / Respiratory Physi

pnoea (Daly, 1984, 1986; Elsner et al., 1966a,b; Whayne andillip, 1967). The respiratory-sympathetic coupling present in

he diving response is strong enough to reverse paroxysmalupraventricular tachycardia (Bisset et al., 1980; Wildenthal et al.,975).

In the experiments of McMullan et al. (2009), only a 30% increasen TE was associated with ADN stimulation. The experiments of

cMullan et al. (2009) were performed in an anesthetized rathereas in our studies the rats were decerebrate. The difference

etween stimuli may also be afactor in the different results. Weelivered a pressure pulse directly to the arterial system thatffected all of the baroreceptors, In contrast, McMullan et al., testedTE baroresponse by pharmacologic agents (phenylepherine andngiotension II) as well as by ADN stimulation and definitely didot observe a robust prolongation of expiration. As discussed laterhe role of anesthetics may be critical in evoking the response of arolongation in Te.

Expiratory brainstem activity sensitive to baroreceptor hasot been found in anesthetized rats (Kanjhan et al., 1995) butas been reported in anesthetized (Lindsey et al., 1998) andecerebrate (Dick and Morris, 2004; Dick et al., 2005) cats. Inentobarbital-anaesthetized rats, BötC neurones were recordednd identified on the basis of location, phasic expiratory activity,nd proprio-bulbar axons. None (n = 29) of the recorded neuronesad systole-triggered histograms with a feature indicating sen-itivity to baroreceptor input nor a response to a baro-stimulushat consisted of three current pulses delivered to ADN (Kanjhant al., 1995). Contrasting this in Dial-urethane anaesthetized cats,ix of seven ventrolateral medullary post-I neurones increasedheir firing rate during baroreceptor stimulation and their increasesere associated with TE prolongation. Similarly in decerebrate

ats, nearly half (96/200) of the respiratory modulated neuronesad arterial-pulse modulated activity (Dick and Morris, 2004).

n this and a subsequent study (Dick et al., 2005), expiratoryctivity was preferentially modulated with arterial pulse pres-ure.

The obvious differences in these studies were anesthetic andpecies, however, these are unsatisfying explanations. Anotherossibility may be related to an observation we made in thatulse-modulated activity was grouped in certain ensemble record-

ngs in which the animal had a high pulse pressure. Thus, theagnitude and prevalence of pulse-modulated activity may befunction of the magnitude of the pulse pressure. Indeed, the

ercentage of simultaneously recorded neurones exhibiting pulse-odulated activity was significantly correlated with pulse pressure

Dick and Morris, 2004). Despite this correlation the raw recordsrom Kanjhan et al. have a robust (appearing to range from 30 to0 mm Hg) arterial pulse pressure whereas the perfused rat has ainimal pulse pressure (but the transient increases in perfusion

ressure are approximately 60 mm Hg thereby mimicking largeressure pulses). In summary we have a plausible explanation aso why Kanjhan et al. (1995) did not find baromodulated activity inötzinger Complex activity in rats.

.3. Computational model

Our model represents a first attempt to integrate sympatho-espiratory circuits. The model clarifies the role of baroreceptorsn activating post-I neurones and thereby indirectly inhibitingug-E neurones and demonstrates that excitatory synaptic driveo the post-I neurones can account for the prolongation in

xpiration.

The model focuses on the interaction between BötC and RVLM.he primary cell types in the BötC are inhibitory although excitatoryost-I neurones may exist and may be responsible for post-I modu-

atory projections to the pons and for upper airway motor control.

& Neurobiology 174 (2010) 135–145 143

Therefore, future modelling could be based on VRC interactionwith the RVLM/CVLM using both inhibitory and excitatory connec-tions. For example, should excitatory post-I neurones project to theCVLM and in turn these CVLM neurones inhibit RVLM activity, thiswould produce the same result as the current model shows. Also, asmentioned above, our current model does not account for the dif-ferent respiratory-modulated patterns observed in CVLM neurones(Mandel and Schreihofer, 2006). Clarification of the respiratory net-work interactions with the RVLM and CVLM will be in focus of ourfuture modelling studies.

Post-I neurones in our model display a reduction in the rate ofdecline in firing frequency, and prolongation of their discharge thatprovides an increase in expiratory duration (Feldman and Cohen,1978; Hayashi et al., 1996; Krolo et al., 2005; Manabe and Ezure,1988; Parkes et al., 1994). Our model contains a special populationof baro-sensitive cells in the NTS projecting to the post-I neu-rones, whose activity is controlled centrally (in the current modelby early-I(2) population of rVRG). This population could be P-cellsas they are activated in the baroreflex (Rogers et al., 1993). Inter-estingly, the model is consistent with the control of the NTS pump(P) cells receiving early-inspiratory inhibition previously describedby Miyazaki et al. (1999). Because stimulation of pulmonary affer-ents activates post-I neurones via P cells of NTS (Hayashi et al.,1996), P cells may be involved in both the Hering–Breuer reflexand baro-respiratory response. Yet another possibility for controlof the gain of baroreflex at the level of NTS could be if early inspira-tory inhibition comes from the pons (see Fig. 2). Indeed, electricalstimulation of parabrachial nucleus suppresses the gain of carotidsinus afferent input to NTS (Felder and Mifflin, 1988), so the con-trol of baroreflex gain can be modelled by incorporating inhibitoryinspiratory population to the pontine compartment projecting tothe second-order baro-sensitive cells in the NTS that excite post-Ineurones.

4.4. Two pathways of the sympathetic baroreceptor reflex in thebrainstem

The results of our experimental and modelling studies supportthe proposed concept (Fig. 2) that the sympathetic barorecep-tor reflex, providing negative feedback from baroreceptors to therostral ventrolateral medulla and SNA, has two pathways. Thefirst, direct path leads from the baroreceptor neurones in NTSto CVLM which inhibits RVLM hence lowering SNA (Dampney,1994; Guyenet, 1990). The second pathway is mediated by respi-ratory circuits, specifically by the post-I neurones of BötC whichinhibit RVLM and prolong expiration. This baroreflex pathwayis strongly dependent on the respiratory–sympathetic interac-tions and requires an intact pons to be expressed. This concept,similar to that developed by Guyenet and Koshiya for the sympa-thetic chemoreflex (Guyenet, 2000; Guyenet and Koshiya, 1995;Koshiya and Guyenet, 1996) requires further experimental inves-tigations.

Acknowledgements

We gratefully acknowledge the support of NIH grants R33HL087377, NS057815, HL090554, and HL033610; American HeartAssociation, SDG 073503N; and the British Heart Foundation. DavidBaekey was also funded by a Leverhulme Fellowship from the Uni-versity of Bristol. J.F.R.P. was in receipt of a Royal Society Wolfsom

Research Merit Award.

Appendix A

See Table A.1.

144 D.M. Baekey et al. / Respiratory Physiology

Table A.1Weights of synaptic connections in the network.

Target population(location)

Excitatory drive {weight of synaptic input} or sourcepopulation {weight of synaptic input from singleneuron}

ramp-I(rVRG)

drive(pons) {2.0};early-I(2) {−0.3}*; pre-I/I{0.06}; aug-E{−0.1}*; post-I{−2.0}*.

early-I(2)(rVRG)

drive(pons) {2.5}*;aug-E {−0.25}; post-I {−0.5}*.

pre-I/I(pre-BötC)

drive(raphe) {0.3}; drive(RTN) {0.22}*; drive(pons){0.65}*;pre-I/I {0.03}; aug-E {−0.06}*; post-I {−0.16}*.

early-I(1)(pre-BötC)

drive(RTN) {1}*; drive(pons) {1.1};pre-I/I {0.1};* aug-E {−0.265}*; post-I {−0.45}*.

aug-E(BötC)

drive(RTN) {1.5}*; drive(pons) {1.2}*;early-I(1) {−0.135}*; post-I {−0.3}.

post-I(BötC)

drive(RTN) {0.05}*; drive(pons) {1.65}*;early-I(1) {−0.025}*; aug-E {−0.01}; 2◦ BaroP){0.075}*.

post-I(e)(BötC)

drive(RTN) {0.05}*; drive(pons) {1.65}*;early-I(1) {−0.025}*; aug-E {−0.01}; P-cells {0.075}.

CVLM*(VLM)

2◦ Baro {0.05}*.

RVLM*(VLM)

drive(RTN) {1}*;early-I(2){−0.01}*; post-I{−0.05}*; CVLM{−0.025}*;IE{0.05}*.

IE*(PONS)

ramp-I {0.2}*; post-I {0.35}*.

2◦ Baro P)*NTS)

stim*# {1};early-I(2) {−0.05}*.

2◦ Baro*(NTS)

stim*# {1}.

Values in brackets represent relative weights of synaptic inputs from the corre-sponding source populations wji or drives wdmi .*Populations not present in the model of Smith et al. (2007) and weights of connec-t#

ig

R

B

B

B

B

B

C

C

D

D

D

D

D

D

D

D

ions adjusted in the present model relative to that model.Stimulus representing the perfusion pressure changes PP is modelled as anntegrated rectangular impulse barostimulus 400 ms long of unity height with inte-ration time constants 500 ms up and 2000 ms down.

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